Work in this group is aimed at elucidating the mechanism of stimulus response coupling mediated by Ca2+ and calmodulin using the calmodulin-stimulated protein phosphatase, calcineurin, as a model system. Two major goals are 1) to elucidate the crystal structure of the calmodulin-calcineurin complex and to characterize the conformational changes induced by Ca2+ binding to this complex, and 2) to determine the molecular basis for the specific interactions of the immunosuppressive drugs, FK506 and cyclosporin A, complexed with their respective immunophillins, with calcineurin. During the past year, Hao Ren has devoted his efforts to reconstitution of the BETA-isoform of calcineurin A, and its calmodulin-independent derivative from the two subunits expressed in E. coli. Reconstitution of the two forms of the enzyme requires calcineurin B and Mn2+ and is facilitated by calmodulin. In the case of native calcineurin 10 to 20 mg of highly purified calcineurin is produced per liter of culture medium. The calcineurin B-binding domain of calcineurin A was mapped to residues 341 to 386. The determination of the structure of a complex of calcineurin B with a synthetic calcineurin B-binding peptide corresponding to this region is presently under way in collaboration with Drs. Adriaan Bax and Jacob Anglister (NIDDK). Also, in collaboration with Dr. Bax and his colleagues, we determined the solution structure of Ca2+-free calmodulin thus allowing the identification of the nature of the Ca2+-dependent conformational changes.